Giardia is a widespread zoonotic intestinal parasite and is one of the ten major parasites of humans, causing significant acute and chronic diarrheal disease. Giardia is a global health concern, yet fundamental biological questions remain, and we know little about in vivo infection dynamics and pathogenesis. Recent anti-giardial drug development strategies that have focused on high throughput screening of large compound libraries to identify new candidate drugs have met with limited success. In addition to metabolism, flagellar motility, attachment and cell division are critical for host colonization. Therapies that target these processes could interfere with trophozoite colonization and proliferation, reducing the length or severity of the infection, and reducing the number of shed cysts. In the R21 phase we will develop more effective tools for drug target identification and validation. Specifically, we will create bioreporter-based in vitro and in vivo assays to monitor and quantify specific cellular processes. We will then infect mice with bioreporter strains and use non-invasive in vivo optical imaging to assay parasite cellular processes over the course of an infection (Aim 1). To aid in target identification, we also will develop and use CRISPR-based methods of genome editing to create knockout and knockdown mutants in Giardia (Aim 2). Using methods we have developed in the R21 phase, we will assess and prioritize cellular processes and possible drug targets. Specifically, we will create mutants in key parasitic processes using bioreporter strains to assay mutant phenotypes in vitro (Aim 3) and in vivo (Aim 4). Using new genetic tools (Aim 2), we will create several mutants in motility, cell division, metabolism or encystation. Mutants will be used to infect mice, and we will use our optical imaging assays to evaluate temporal and spatial defects in parasite proliferation and encystation (Aim 4). These methods are essential for identifying drug targets, evaluating the effects of anti-giardial drug candidates, and for validating candidate efficacy and toxicity.